Multiplex Q-PCR Arrays

a q-pcr array and array technology, applied in the field of multi-dimensional q-pcr arrays, can solve the problems of affecting the replication process, and the number of different nucleic acid sequences that can be simultaneously amplified and detected is very limited, and achieves the obstacle of achieving a truly multiplexed q-pcr

Active Publication Date: 2008-07-24
CALIFORNIA INST OF TECH
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Benefits of technology

[0011]One aspect of the invention is a method comprising; performing a nucleic acid amplification on two or more nucleotide sequences to produce two or more amplicons in a fluid wherein the array comprises a solid surface with a plurality of nucleic acid probes at independently addressable locations; and measuring the hybridization of the amplicons to the two or more nucleic acid probes while th

Problems solved by technology

Nevertheless, the number of different nucleic acid sequences that can be simultaneously amplified and detected is very limited.
However, practical issues affect the replication process adversely and the efficiency of PCR, defined as the probability of generating a replica of each template molecule, is usually smaller than the desired factor of two.
Attempts at employing QPCR for the simultaneous amplification and detection of many target amplicons in a single well are plagued with practical issues that present obstacles to achieving a truly multiplexed Q-PCR.
This type of sample splitting reduces the amount of material in each individual amplification well, creating issues of sample size, and necessitating precise sample distribution across the wells.
This limita

Method used

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Examples

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example 1

[0247]This example demonstrates the use of a real-time microarray to measure the binding of multiple analytes to multiple probes.

[0248]FIG. 15 shows the layout of a 6×6 DNA microarray. Three different DNA probes (1, 2, and Control) with three different concentrations (2 μM, 10 μM, and 20 μM) are spotted and immobilized on the surface as illustrated. The probes contain a single Cy3 fluorescent molecule at the 5′ end. The DNA targets in this experiment contain a quencher molecule. The analyte binding in this system results in quenching of fluorescent molecules in certain spots. FIG. 16 shows a few samples of the real-time measurements of the microarray experiment wherein the control targets are added to the system. As illustrated in FIG. 16, the spots are quenched due to analyte binding.

[0249]FIGS. 17-20 each show data for 4 different spots with similar oligonucleotide capturing probes. The target DNA analyte is introduced in the system at time zero and quenching (reduction of signal)...

example 2

[0251]This example provides a derivation of an algorithm, and the use of the algorithm to determine analyte concentration, such as amplicon concentration, from a real-time binding data. The derivation proceeds as follows:

[0252]Assume that the hybridization process starts at t=0, and consider discrete time intervals of the length Δt. Consider the change in the number of bound target molecules during the time interval (iΔt, (i+1)Δt). We can write

nb(i+1)−nb(i)=[nt−nb(i)]pb(i)Δt−nb(i)pr(i)Δt,

where nt denotes the total number of target molecules, nb(i) and nb(i+1) are the numbers of bound target molecules at t=iΔt and t=(i+1)Δt, respectively, and where pb(i) and pr(i) denote the probabilities of a target molecule binding to and releasing from a capturing probe during the ith time interval, respectively. Hence,

nb(i+1)-nb(i)Δt=[nt-nb(i)]pb(i)-nb(i)pr(i).(1)

[0253]It is reasonable to assume that the probability of the target release does not change between time intervals, i.e., pr(i)=pr, fo...

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Abstract

This invention provides methods and systems for measuring the concentration of multiple nucleic acid sequences in a sample. The nucleic acid sequences in the sample are simultaneously amplified, for example, using polymerase chain reaction (PCR) in the presence of an array of nucleic acid probes. The amount of amplicon corresponding to the multiple nucleic acid sequences can be measured in real-time during or after each cycle using a real-time microarray. The measured amount of amplicon produced can be used to determine the original amount of the nucleic acid sequences in the sample.

Description

CROSS-REFERENCE[0001]This application claims priority to U.S. Provisional Application No. 60 / 834,051, filed Jul. 28, 2006, which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]Nucleic acid target amplification assays such as polymerase chain reaction process (PCR), in principle, amplify and replicate specific sequences of nucleic acids of a DNA template in vitro. These assays have become a powerful tool in molecular biology and genomics, since they can increase the number of copies of target molecules with great specificity. It is of great interest to efficiently multiplex the amplification process and thus allow for multiple target amplification and quantification.[0003]Currently, various homogeneous (closed-tube) assays are available for PCR. These assays detect the target amplicons (i.e., Quantitative PCR or QPCR). Nevertheless, the number of different nucleic acid sequences that can be simultaneously amplified and detected is very limited. T...

Claims

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Application Information

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IPC IPC(8): C40B30/04C40B30/00C12M1/00C12Q1/68
CPCC12Q1/6851C12Q2565/519C12Q2561/113C12Q1/686G01N21/6408G01N21/6428G01N2021/6432G01N2021/6439
Inventor HASSIBI, ARJANGHASSIBI, BABAKVIKALO, HARIS
Owner CALIFORNIA INST OF TECH
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